The invention relates to novel inhibitors of urokinase for treating malignant tumors and metastasis.
The dissemination and metastasis of solid tumors in surrounding tissue is made possible by their ability to break down the extracellular matrix in the environment of the tumor cell and/or penetrate the basal membrane. Aside from various matrix metalloproteinases and cathepsins, the plasminogen activator urokinase (uPA) is, in particular, of central importance in this process (P. Mignatti and D. B. Rifkin, Physiol. Rev. 73, 161-195, 1993). Thus, uPA activates plasminogens; the resulting plasmin is able to break down the components of the extracellular matrix (fibrin, fibronectin, laminin and proteoglycans inter alia) and also activate metalloproteases and prourokinase to give uPA (U. Reuning et al., Int. J. Oncol. 13, 893-906, 1998). Both prourokinase and uPA bind to the uPA receptor (uPAR), which is a specific receptor located on the cell surface. This results in the activity of uPA, and consequently plasminogen activation, being augmented and focused in the direct environment of the tumor cell. The importance of this cell-associated plasminogen activator system for tumor growth and tumor dissemination has been demonstrated both in cell-biological studies and in animal models. Thus, the invasive potential of tumor cells is diminished by inhibiting the enzymatic activity of uPA with the natural inhibitors PAI-1 and PAI-2 (J.-F. Cajot et al., Proc. Natl. Acad. Sci. USA 87, 6939-6943, 1990; M. Baker et al., Cancer Res. 50, 4876-4684, 1990). In chick embryos, the formation of lung metastases caused by human carcinoma cells was almost completely inhibited by adding antibodies directed against uPA (L. Ossowski et al., Cell 35, 611-619, 1983).
In recent years, the clinical relevance of the factors involved in the plasminogen activator system (uPA, uPAR, PAI-1 and PAI-2) for the prognosis of patients who have solid malignant tumors has been intensively investigated. In particular, the content of uPA in the tissue of various tumors has been found to be a prognosis factor. Thus, patients who have a high uPA level have a poorer prognosis than do those who have a low uPA concentration in the tumor (M. Schmitt et al., Thromb. Haemost. 78, 285-296, 1997; R. W. Stephens et al., Breast Cancer Res. Treat. 52, 99-111, 1998). An elevated concentrations of uPAR in the tumor tissue is also correlated with a poor prognosis (H. Pedersen et al., Cancer Res. 54, 4671-4675, 1994; C. Duggan et al., Int. J. Cancer 61, 597-600, 1995).
From the findings regarding the prognostic value of the uPA content and uPAR content in tumor tissue, it can be assumed that synthetic uPA inhibitors are able to suppress the invasion and dissemination of tumor cells. However, the number of uPA inhibitors which are known thus far is relatively small. The majority possess only slight specificity and potency, as is the case for various benzamidine and xcex2-naphthamidine derivatives (J. Stxc3xcrzebecher and F. Markwardt, Pharmazie 33, 59-602, 1978). While the amiloride which is described by Vassalli and Belin (FEBS Letters 214, 187-191, 1997) as being a uPA inhibitor is a specific inhibitor of uPA, the inhibition is only weak (ki=7 xcexcM).
4-Substituted benzothiophene-2-carboxamidines have been found to be more active uPA inhibitors (Ki=0.16 xcexcM in the case of compound 623). Inhibitors of this type also inactivate uPA which is bound to uPAR (M. J. Towle et al., Cancer Res. 53, 2553-2559, 1993). The benzothiophene derivatives are very specific and they only have a low inhibitory effect on plasmin and tissue-type plasminogen activator (tPA); however, it is a very elaborate matter to synthesize compounds of this type.
While 4-aminomethylphenylguanidine derivatives have a comparable specificity, their inhibitory effect on uPA (Ki=2.4 xcexcM for the most active compound) is comparatively low (S. Sperl et al., Proc. Natl. Acad. Sci. USA 97, 5113-5118, 2000).
In contrast to this, Nxcex1-triisopropylphenylsulfonyl-3-amidinophenylalanine derivatives achieve micromolar Ki values (0.41 xcexcM in the case of the most active compound); however, they are very nonspecific uPA inhibitors, having the same or a stronger inhibitory effect on trypsin, thrombin and plasmin (J. Stxc3xcrzebecher et al., Bioorg. Med. Letters 9, 3147-3152, 1999). WO 99/05096 discloses improved xcex2-naphth-amidines which are very effective uPA inhibitors. While this patent reports IC50 values in the nanomolar range, it provides no data with regard to selectivity and biological activity.
Thus far, only a few peptides which are derived from the substrate sequence have been reported to be uPA inhibitors. Kettner and Shaw (Methods in Enzymology, 80, 826-842, 1981) described chloromethyl ketones which, while inhibiting uPA irreversibly, are not suitable for in-vivo use.
EP 18 32 71 discloses lysine derivatives which inhibit uPA to a certain degree; however, they also inhibit other comparable enzymes and can consequently only be used very specifically, or in a restricted manner, for medical purposes. The same applies to the low molecular weight polypeptides (approx. 50 amino acids) which are reported in WO 95/17885 to be uPA inhibitors and which are derived from natural inhibitors. Their peptide nature, and their molecular size, greatly restrict their in-vivo use.
However, WO 00/05245 has very recently reported peptidyl aldehydes which contain an argine C-terminally and a D-serine in P3 and which effectively inhibit uPA. However, the aldehyde function gives rise to instability and low selectivity. After the Ser hydroxyl had been acylated, the key compound iBuOCO-D-Ser-Ala-Arg-H was observed to have a relative bioavailability of 87% following s.c. administration (S. Y. Tamura et al., Bioorg. Med. Chem. Lett. 10, 983-987, 2000). Furthermore, notable advances, with regard to both the inhibitory effect and the bioavailability, were achieved when using tripeptide derivatives of the D-Phe-Pro-Arg type in the search for inhibitors of thrombin, an enzyme which is related to uPA, when agmatine, trans-4-aminomethylcyclohexylamine or 4-amidinobenzylamine was incorporated C-terminally. Picomolar Ki values were achieved and the oral bioavailability was improved (T. J. Tucker et al., J. Med. Chem. 40, 1565-1569 and 3687-3693, 1997); however, no uPA inhibitors were found. Thus, while melagatran, which possesses a 4-amidinobenzylamide residue C-terminally, inhibits trypsin (Ki=2.0 nM) and thrombin (Ki=2.0 nM) very nonspecifically, its inhibition of uPA, with a Ki=6.3 xcexcM, is three orders of size weaker (D. Gustafsson et al., Blood Coagul. Fibrinolysis 7, 69-79, 1996; WO 94/29336).
The invention is based on the object of specifying an active compound which inhibits urokinase with high activity and specificity, which can be prepared by means of a synthesis which is as uncomplicated as possible, and which is also suitable for therapeutic applications.
Surprisingly, it has been found that acylated amidino-benzylamine in accordance with the formula I cited in patent claim 1, in particular compounds of 4-amidinobenzylamine in which X, R1, R2 and R3 give natural and/or unnatural amino acids, inhibit urokinase very effectively and selectively. In this connection, amidinobenzylamine forms a particularly active urokinase inhibitor if the amidino group is in the 4 position, Gly and D-Ser are bonded as amino acids and the compound possesses an N-terminal protecting group R4 which is composed of an arylsulfonyl radical or aralkylsulfonyl radical.
Esters, in particular those with oxycarboxylic acids, can be employed as prodrugs if they are hydrolyzed during the course of enteral uptake. It has also been found, surprisingly, that some of these oxycarbonyl derivatives of the compounds according to the invention are also very strong urokinase inhibitors.
Aside from urokinase, the glycine derivatives inhibited other enzymes to a markedly lesser degree, which means that these amidinobenzylamine derivatives according to the invention constitute a novel group of highly active and very selective uPA inhibitors. By contrast, compounds which do not carry any H as R1 (e.g. alanine derivatives) no longer inhibit urokinase selectively but are also strong inhibitors of trypsin, thrombin and plasmin.
As a rule, the compounds are present as salts with mineral acids, preferably as hydrochlorides, or as salts with suitable organic acids.
The compounds of the formula I can be prepared in a relatively simple manner using known methods, as described below:
The starting compound 4-cyanobenzylamine is prepared by Gabriel synthesis (G. Wagner and I. Wunderlich, Pharmazie 32, 76-77, 1977; B. C. Bookser and T. C. Bruice, J. Am. Chem. Soc. 113. 4208-4218, 1991) from 4-cyanobenzyl bromide. The Boc-protected acetyloxamidinobenzylamine is obtained from the 4-cyanobenzylamine which has been prepared in this way. The other amino acids and the R4 protecting group are coupled on employing standard coupling methods and using Boc as the N-terminal protecting group. The second amino acid can also be coupled directly as an N-arylsulfonyl- or N-aralkylsulfonyl-protected amino acid. The peptide analogs are synthesized sequentially, beginning with the acetyloxamidinobenzylamine. In order to synthesize the corresponding esters, the target compound is reacted with the corresponding acid chloride. Most of the products crystallize well and can be readily purified in this way. In the final step, the inhibitors are purified by means of preparative, reversed-phase HPLC.
The invention will be explained in more detail below with the aid of two implementation examples: